Zhao Kaitao, Wang Jingjing, Wang Zichen, Wang Mengfei, Li Chen, Xu Zaichao, Zhan Qiong, Guo Fangteng, Cheng Xiaoming, Xia Yuchen
State Key Laboratory of Virology and Biosafety and Hubei Province Key Laboratory of Allergy and Immunology, Institute of Medical Virology, TaiKang Medical School, Wuhan University, Wuhan, China.
Wuhan University Center for Pathology and Molecular Diagnostics, Zhongnan Hospital of Wuhan University, Wuhan, China.
PLoS Pathog. 2025 Jan 3;21(1):e1012824. doi: 10.1371/journal.ppat.1012824. eCollection 2025 Jan.
Chronic hepatitis B virus (HBV) infection can significantly increase the incidence of cirrhosis and liver cancer, and there is no curative treatment. The persistence of HBV covalently closed circular DNA (cccDNA) is the major obstacle of antiviral treatments. cccDNA is formed through repairing viral partially double-stranded relaxed circular DNA (rcDNA) by varies host factors. However, the detailed mechanisms are not well characterized. To dissect the biogenesis of cccDNA, we took advantage of an in vitro rcDNA repair system to precipitate host factors interacting with rcDNA and identified co-precipitated proteins by mass spectrometry. Results revealed the MRE11-RAD50-NBS1 (MRN) complex as a potential factor. Transiently or stably knockdown of MRE11, RAD50 or NBS1 in hepatocytes before HBV infection significantly decreased viral markers, including cccDNA, while reconstitution reversed the effect. Chromatin immunoprecipitation assay further validated the interaction of MRN complex and HBV DNA. However, MRN knockdown after HBV infection showed no effect on viral replication, which indicated that MRN complex inhibited the formation of cccDNA without affecting its stability or transcriptional activity. Interestingly, Mirin, a MRN complex inhibitor which can inhibit the exonuclease activity of MRE11 and MRN-dependent activation of ATM, but not ATM kinase inhibitor KU55933, could decrease cccDNA level. Likewise, the MRE11 endonuclease activity inhibitor PFM01 treatment decreased cccDNA. MRE11 nuclease assays indicated that rcDNA is a substrate of MRE11. Furthermore, the inhibition of ATR-CHK1 pathway, which is known to be involved in cccDNA formation, impaired the effect of MRN complex on cccDNA. Similarly, inhibition of MRE11 endonuclease activity mitigated the effect of ATR-CHK1 pathway on cccDNA. These findings indicate that MRN complex cooperates with ATR-CHK1 pathway to regulate the formation of HBV cccDNA. In summary, we identified host factors, specifically the MRN complex, regulating cccDNA formation during HBV infection. These findings provide insights into how HBV hijacks host enzymes to establish chronic infection and reveal new therapeutic opportunities.
慢性乙型肝炎病毒（HBV）感染可显著增加肝硬化和肝癌的发病率，且尚无治愈性治疗方法。HBV共价闭合环状DNA（cccDNA）的持续存在是抗病毒治疗的主要障碍。cccDNA是通过多种宿主因子修复病毒部分双链松弛环状DNA（rcDNA）形成的。然而，具体机制尚未完全明确。为了剖析cccDNA的生物发生过程，我们利用体外rcDNA修复系统沉淀与rcDNA相互作用的宿主因子，并通过质谱鉴定共沉淀蛋白。结果显示MRE11-RAD50-NBS1（MRN）复合物是一个潜在因子。在HBV感染前，瞬时或稳定敲低肝细胞中的MRE11、RAD50或NBS1可显著降低包括cccDNA在内的病毒标志物水平，而恢复表达则可逆转该效应。染色质免疫沉淀试验进一步验证了MRN复合物与HBV DNA的相互作用。然而，在HBV感染后敲低MRN对病毒复制无影响，这表明MRN复合物抑制cccDNA的形成，但不影响其稳定性或转录活性。有趣的是，Mirin是一种MRN复合物抑制剂，可抑制MRE11的核酸外切酶活性以及MRN依赖的ATM激活，但ATM激酶抑制剂KU55933则无此作用，Mirin可降低cccDNA水平。同样，MRE11核酸内切酶活性抑制剂PFM01处理也可降低cccDNA水平。MRE11核酸酶分析表明rcDNA是MRE11的底物。此外，已知参与cccDNA形成的ATR-CHK1通路受到抑制会削弱MRN复合物对cccDNA的作用。同样，抑制MRE11核酸内切酶活性可减轻ATR-CHK1通路对cccDNA的影响。这些发现表明MRN复合物与ATR-CHK1通路协同调节HBV cccDNA的形成。总之，我们鉴定出了在HBV感染期间调节cccDNA形成的宿主因子，特别是MRN复合物。这些发现为HBV如何劫持宿主酶建立慢性感染提供了见解，并揭示了新的治疗机会。
